Various methods of braking the diffraction limit in microscopy have been developed in the state of the art. A method abbreviated to PALM (photo activated light microscopy) is known from WO 2006/127692 or DE 102006021317 A1, which uses a label substance for imaging a specimen which can be activated e.g. by means of optical radiation. Only in the activated state can the label substance emit specific fluorescence radiation. Non-activated molecules of the label substance emit no, or at least no noticeable, fluorescence radiation even after irradiation by excitation radiation. The activation radiation is therefore generally called a switching signal. In the PALM method, the switching signal is applied such that at least some of the activated label molecules are spaced apart from neighbouring activated molecules such that these label molecules are isolated, as measured by the optical resolution of the microscopy, or can be isolated subsequently by image processing methods. It is said that a sub-set of isolated fluorescence emitters is formed. After recording the fluorescence radiation, for these isolated emitters the centre of their radiation distribution is then identified, which distribution is a result, of the limited optical resolution. Doing this, the position of the molecules can be determined computationally with higher precision than the optical resolution itself allows. This procedure is called localization. The increased resolution so by computational determination of the centre of the diffraction distribution is also called “superresolution” in the technical literature. It requires that in the specimen at least a sub-set of the activated label molecules is distinguishable with the optical resolution, i.e. consists of isolated emitters. Their position can be determined with higher precision, then; they can be localized.
To isolate individual label molecules, the PALM principle utilizes statistical effects. In the case of a label molecule which can be excited to fluorescence radiation after receiving the switching signal of given intensity, it can be ensured, by adjusting the intensity of the switching signal, that the probability of activating label molecules present in a given area of the specimen is so small that there are enough sub-sections in which only label molecules emit fluorescence radiation which molecules are distinguishable within the optical resolution.
The PALM principle was refined with respect to the activation of the molecules to be detected. Thus for example, in the case of molecules that have as long-living non-fluorescing and a short-living fluorescing state, a separate activation needing activation radiation differing spectrally from the excitation radiation is not necessary. Instead the specimen is first activated with high-intensity illumination radiation such that the vast majority of the molecules is converted to the long-living state (e.g. a triplet state) in which they cannot fluoresce. The remaining, still fluorescing molecules are thereby isolated in respect of the optical resolution.
It may also be noted that the PALM principle has meanwhile also acquired other abbreviations, such as for example STORM, in the technical literature. In this description, the abbreviation PALM is used for all microscopy imagings which achieve a spatial resolution beyond the optical resolution of the apparatus used by first isolating and then localizing fluorescence molecules. The PALM method has the advantage that no high spatial resolution is required for the illumination. A simple widefield illumination is possible.
The PALM principle requires many frames of the specimen to be recorded, each of which contains a sub-set of isolated molecules. In order to image the specimen completely, the quantity of all frames must ensure that if possible all molecules were contained at least once in a sub-set. The PALM method therefore usually requires a large number of frames, which means that it takes a certain period of time to record a complete image. This is associated with a considerable computational outlay as a large number of molecules must be computationally localized in each frame. Large amounts of data are accumulated.
There is now a need not only to record high-resolution images in is colour channel, but to obtain colour information, i.e. an indication of the wavelength of the fluorescing emitters. In fluorescence microscopy, the state of the an knows a microscope according to FIG. 8. The fluorescence microscope 100 shown there comprises an illumination beam path 3 as well as an imaging beam path 4 which illuminate a specimen 2 with excitation radiation via a common objective 5 and image the fluorescing specimen 2. The illumination beam path 3 is combined with the imaging beam path 4 via a beam splitter 6, usually dichroic, such that illumination radiation from the illumination beam path 3 is incident on the specimen 2 through the objective 5 as well as the imaging of the specimen is carried out through the objective 5 and via the imaging beam path 4. The illumination beam path 3 usually has several spectral channels; in the representation in FIG. 8 two laser sources L1 and L2 are shown by way of example, the radiation of which is combined via a beam splitter 5. The illumination beam path 3 thus illuminates the specimen 2 with radiation of at least two wavelengths, with the result that a multicoloured excitation of the specimen 2 to fluorescence radiation is effected. The specimen 2 also emits multicoloured fluorescence radiation (naturally this could also be the case for as monochrome fluorescence excitation and different fluorescence molecules). In the imaging beam path 4, the image of the specimen 2 is therefore divided onto three colour channels, i.e. directed to three cameras K1, K2 and K3, via two beam splitters 8 and 9 as well as suitable lens systems not described in more detail. The splitting via the beam splitters 8 and 9 effects a spectrally selective split to the cameras K1 to K3. Alternatively or in addition, suitable colour filters can be used. Several colour channels are thus obtained, one for each camera. However, a disadvantage of this design is that the camera systems used are very expensive due to the high resolution required. Furthermore, the installation space for the microscope 100 is large on account of the required beam paths and colour splitters. The cameras are also usually cooled and likewise require a large installation space, further problem is that the cameras K1, K2 and K3 must be aligned precisely relative to one another so that the images of the individual colour channels are subsequently positioned correctly relative to one another. Any alignment error between the beam paths of the individual colour channels would result in a colour aberration constituting a chromatic aberration in the complete image.
It would be conceivable to use the microscope 100 of FIG. 8 for the PALM principle, but then the amount of data accumulating as well as the computational outlay would be multiplied by the number of colour channels.